This invention relates to methods for making and using and compositions containing Epstein Barr virus (EBV) gp350 DNA and protein sequences.
Epstein-Barr virus (EBV), a member of the herpesvirus group, causes infectious mononucleosis in humans. The disease affects more than 90% of the population. Health analysts estimate the cost of the disease in the United States is 100 million dollars per year. The virus is spread primarily by exchange of saliva from individuals who shed the virus. Children infected with EBV are largely asymptomatic or have very mild symptoms, while adolescents and adults who become infected develop typical infectious mononucleosis, characterized by fever, pharyngitis, and adenopathy. People who have been infected maintain anti-EBV antibodies for the remainder of their lives, and are thus immune to further infection. Currently there is no commercially available EBV vaccine.
In addition to its infectious qualities, EBV has been shown to transform lymphocytes into rapidly dividing cells and has therefore been implicated in several different lymphomas, including Burkitt""s lymphoma and oral hairy leukoplakia. EBV has also been detected in tissue samples from nasopharyngeal tumors. Worldwide it is estimated that 80,000 cases of nasopharyngeal cancer occur and it is more prevalent in ethnic Chinese populations.
Development of a live, attenuated vaccine for EBV has been and still is problematic. Because of the potential oncogenic nature associated with EBV, researchers have been reluctant to use a live vaccine approach. This invention overcomes the problems associated with live vaccine development by creating methods and compositions for a subunit vaccine, that does not require the use of a potentially oncogenic live virus. A subunit vaccine uses one or more antigenic proteins from the virus that will elicit an immune response and confer immunity.
Two of the more important antigenic EBV proteins are glycoprotein(s) gp350/300 and gp220/200 that form part of the viral membrane envelope and allow virus particles to bind to and enter human target cells by interacting with the cellular membrane protein, CD21. See Nemerow, J. Virology 61:1416(1987). They have long been singled out as subunit vaccine candidates but difficulties in obtaining antigenically active protein purified from native sources and low yields from recombinantly produced sources have hampered efforts of researcher and vaccine developers. In the literature these proteins are referred to using a variety of molecular weight ranges (350 or 300 kilodaltons (kD) for one of the proteins and 220 or 200 kDs for the other protein). The gp350 or 300 protein is herein referred to as gp350 protein and the gp220 or 200 protein is herein referred to as gp220 protein. Collectively, both proteins are herein referred to as gp350/220 protein(s).
An alternatively spliced, single gene encodes the gp350/220 proteins and results in the generation of gp350 and gp220 mRNA transcripts; no naturally occurring variations in the gp350/220 gene splice sites are known. The gene produces two expression products, the gp350 and gp220 proteins. The open reading frame for the gp350/220 DNA sequence is 2721 base pairs (bp). The entire reading frame encodes the 907 amino acids of gp350. See U.S. Pat. No. 4,707,358 issued to Kieff (1987). The spliced version of the reading frame covers 2130 bases and translates into gp220 protein, a 710 amino acid sequence. The theoretical molecular weights of gp350 protein and gp220 protein are 95 kD and 70 kD, respectively. The measured molecular weights of expressed gp350 protein and gp220 protein vary but are approximately 350 kilodaltons and 220 kilodaltons (kD), respectively. The extensive glycosylation of the proteins accounts for difference between the predicted and actual molecular weights. In any one cell, both gp350 and gp220 proteins are produced at a molar ratio ranging from about 6:1 to 1:1. For example, in B95-8 cells, which are persistently infected with EBV, the ratio appears to vary but sometimes approaches the 6:1 range. See, Miller, Proc. Natl. Acad. Sci. 69:383(1972).
Similarly, recombinant production of these glycoproteins has heretofore usually resulted in a mixture of gp350 and gp220 protein being produced. Heretodate, the gp350/220 proteins have been expressed in rat pituitary, Chinese hamster ovary VERO (African green monkey kidney) cells, as well as in yeast cells. See, Whang, J. Virol. 61:1796(1982), Motz, Gene 44:353(1986) and Emini, Virology 166:387(1988). A bovine papillomavirus virus expression system has also been used to make gp350/220 proteins in mouse fibroblast cells. See, Madej, Vaccine 10:777(1992). Laboratory and vaccine strains of Vaccinia virus have also been used to express gp 350/220 proteins. Modified recombinant versions of the EBV gp350/220 DNA and protein are known in the art. Specifically, recombinant truncated constructs of the gp350/220 gene lacking the membrane spanning sequence have been made. Such constructs still produce a mixture of the two gp 350 and gp220, but deletion of the membrane spanning region permits secretion of the proteins. See, Finerty, J. Gen. Virology 73:449(1992) and Madej, Vaccine 10:777(1992). Also, various recombinantly produced restriction fragments and fusion proteins comprising various gp350/220 sequences have also been made and expressed in E. coli. See EP Patent Publication 0 173 254 published Jul. 24, 1991.
Accordingly, EBV research relating to gp350/220 heretodate has focused either on obtaining efficient expression of the native gp350/220 sequence or on a modified sequence lacking the transmembrane domain, resulting in a mixture of the two alternate spliced versions of the native or transmembrane lacking protein, or on production of epitopic fragment sequences in xcex2-galactosidase fusion proteins.
Partially purified preparations of gp350/220 are known. See, Finerty, J. Gen. Virology 73:449(1992) (recombinantly produced, partially purified). With respect to native gp350/220 protein, in most instances, the purification procedures resulted in inactivating the antigenicity of the protein, making it unacceptable for use in a subunit vaccine. However, highly purified preparations of antigenically active gp350 protein from native (i.e., non-recombinant) sources have been reported in the scientific literature. See, David, J. Immunol. Methods 108:231(1988). Additionally recombinant vaccine virus expressing gp350/220 protein was used to vaccinate cottontop tamarins against EBV-induced lymphoma. See, Morgan, J. Med. Virology 25:189(1988), Mackett, EMBO J. 4:3229(1985) and Mackett, VACCINES ""86, pp293(Lerner R A, Chanock R M, Brown F Eds., 1986, Cold Spring Harbor Laboratory). However, the viral gp350/220 DNA sequence has not heretofore been engineered so as to enable expression solely of either one of the alternate spliced versions of the gene, thereby enabling and ensuring the production of pure gp350 or gp220 protein. Nor has a recombinant or mutant virus been made that expresses one or the other of the gp350 or gp220 proteins.
Generally, splice sites facilitate the processing of pre-mRNA molecules into mRNA. In polyoma virus, splice sites are required for the efficient accumulation of late mRNA""s. Alteration of the 3xe2x80x2 and 5xe2x80x2 splice sites in polyoma virus transcripts decreased or completely blocked mRNA accumulation. See, Treisman, Nature 292:595(1981). In SV40 virus, excisable intervening sequences facilitate mRNA transport out of the nucleus and mRNA stabilization in the nucleus and because these intron/exon junction sequences facilitate binding of small, nuclear, RNP particles, it is thought that prespliced mRNA""s might fail to associate properly with processing pathways. It has been shown that point mutations at exon/intron splice sites reduce exon/intron cleavage and can disrupt pre-mRNA processing, nuclear transport and stability. See, Ryu, J. Virology 63:4386(1989) and Gross, Nature 286:634(1980).
Therefore, until the present invention, the effect of splice site modification on the functional expression and antigenic activity of the proteins encoded by the EBV gp350/220 sequence was at best unknown and unpredictable.
Additional background literature includes the following. EBV biology and disease is generally reviewed in Straus, Annal of Int. Med. 118:45(1993). A description of the EBV BLLFI open reading frame is found in Baer, Nature 310:207(1984). Descriptions of the Epstein-Barr virus gp350/220 DNA and amino acid sequences are found in articles by Beisel, J. Virology 54:665(1985) and Biggin, EMBO J. 3:1083(1984) and in U.S. Pat. No. 4,707,358 issued to Kieff, et al. (1987). A comparison of DNA sequences encoding gp350/220 in Epstein-Barr virus types A and B is disclosed in Lees, Virology 195:578(1993). Monoclonal antibodies that exhibit neutralizing activity against gp350/220 glycoprotein of EBV are disclosed in Thorley-Lawson, Proc. Natl. Acad. Sci. 77:5307(1980). Lastly, splice site consensus sequences for donor and acceptor splice sites are disclosed in Mount, Nucleic Acids Res. 10:459(1982).
In one aspect this invention provides non-splicing variants of the EBV gp350/220 DNA sequence. The DNA sequences of the invention may include an isolated DNA sequence that encodes the expression of homogeneous gp350 protein. The DNA sequence coding for gp350 protein is characterized as comprising the same or substantially the same nucleotide sequence in FIG. 1 wherein the native nucleotides at the donor and acceptor splice sites are replaced with non-native nucleotides, and fragments thereof. This DNA sequence may include 5xe2x80x2 and 3xe2x80x2 non-coding sequences flanking the coding sequence and further include an amino terminal signal sequence. FIG. 1 illustrates the non-coding sequences and indicates the end of the putative signal sequence with an asterisk. It is understood, however, that the DNA sequences of this invention may exclude some or all of these flanking or signal sequences. The non-splicing variant DNA sequences of the invention are produced by introducing mutations into the FIG. 1 DNA sequence in the donor and acceptor splice sites of the gene encoding gp350/220. This eliminates production of gp220 protein so that only the gp350 protein is produced.
Accordingly, in another aspect the invention comprises homogeneous gp350 proteins, and methods of making the proteins by expression of the non-splicing variant of EBV gp350/220 DNA sequence in an appropriate prokaryotic or eukaryotic host cell under the control of suitable expression control sequence. As the term is used here with respect to gp350 proteins, homogeneous means free or substantially free from gp220 protein. We note that homogeneous gp350 protein, recombinantly produced in mammalian or insect cells, has not to our knowledge ever been reported in the scientific literature heretofore.
In yet another aspect, homogeneous gp350 proteins, additionally having deletions resulting in a secreted product are provided. Such deletions comprise either removal of the transmembrane region or removal of the transmembrane region and the remaining C-terminus of gp350. Such additionally modified DNA sequences and the proteins encoded thereby are yet another aspect of this invention.
Also provided is a recombinant DNA molecule comprising vector DNA and a DNA sequence encoding homogeneous gp350 protein. The DNA molecule provides the gp350 sequence in operative association with a suitable regulatory sequence capable of directing the replication and expression of homogeneous gp350 in a selected host cell. Host cells transformed with such DNA molecules for use in expressing recombinant homogeneous gp350 are also provided by this invention.
The DNA molecules and transformed host cells of the invention are employed in another aspect of the invention, a novel process for producing recombinant homogeneous gp350 protein or fragments thereof. In this process a cell line transformed with a DNA sequence encoding a homogeneous gp350 protein or fragment thereof (or a recombinant DNA molecule as described above) in operative association with a suitable regulatory or expression control sequence capable of controlling expression of the protein is cultured under appropriate conditions permitting expression of the recombinant DNA. The expressed protein is then harvested from the host cell or culture medium by suitable conventional means. The process may employ a number of known cells as host cells; presently preferred are mammalian cells and insect cells.
The DNA sequences and proteins of the present invention are useful in the production of therapeutic and immunogenic compounds having EBV antigenic determinants. Such compounds find use in subunit vaccines for the prophylactic treatment and prevention of EBV related diseases, such as mononucleosis, Burkitt""s lymphoma and nasopharyngeal carcinoma. Accordingly, in yet another aspect the invention comprises such therapeutic and/or immunogenic pharmaceutical compositions for preventing and treating EBV related conditions and diseases in humans such as infectitious mononucleosis, Burkett""s lymphoma and nasopharyngeal carcinoma. Such therapeutic and/or immunogenic pharmaceutical compositions comprise a immunogenically inducing effective amount of one or more of the homogeneous gp350 proteins of the present invention in admixture with a pharmaceutically acceptable carrier such as aluminum hydroxide, saline and phosphate buffered saline as are known in the art. By xe2x80x9cimmunogenically inducingxe2x80x9d we mean an amount sufficient for stimulating in a mammal the production of antibodies to EBV. Alternatively, the active ingredient may be administered in the form of a liposome-containing aggregate. For prophylactic use, such pharmaceutical compositions may be formulated as subunit vaccines for administration in human patients. Patients may be vaccinated with a dose sufficient to stimulate antibody formation in the patient; and revaccinated after six months or one year.
A further aspect of the invention therefore is a method of treating EBV related diseases and conditions by administering to a patient, particularly to a human patient, an immunogenically inducing therapeutically effective amount of a homogeneous gp350 protein in a suitable pharmaceutical carrier. Still another aspect of the invention is a method of stimulating an immune response against EBV by administering to a patient an immunogenically inducing effective amount of a homogeneous gp350 protein in a suitable pharmaceutical vehicle.
Other aspects and advantages of the invention are described further in the following detailed description.